Aim 1. Regulation of axonal recruitment of degradative lysosomes in healthy and diseased neurons. Efficient degradation of misfolded protein aggregates and dysfunctional organelles by lysosomes maintains cellular homeostasis essential for neuron function and survival. Lysosome deficits are implicated in several major neurodegenerative diseases. Genetic defects in lysosomal hydrolases and membrane proteins result in neuropathology in neurological lysosomal-storage diseases (LSDs). With these clinical implications, there is growing interest in understanding the causal relationship between an impaired lysosome system and pathogenesis. While enzymatically active degradative lysosomes are enriched in the cell body, lysosome-like organelles are also found in distal axons, where their distribution and motility patterns have not been well characterized. The impact of lysosome positioning and degradation capacity on axonal physiology and pathology remains elusive. Our recent study revealed that LAMP1 is distributed amongst a heterogeneous population of endocytic and lysosomal organelles and is therefore not a reliable and specific marker for assessing distribution of degradative lysosomes in neurons, nor a sensitive indicator to reveal the pathological response of lysosomes to disease conditions in vivo (Cheng et al., JCB 2018). Thus, applying fluorescent probes that selectively bind to active lysosomal enzymes would be ideal for detecting mature degradative lysosomes in axons. We used activity-based fluorescent probes to label three lysosomal hydrolases: cathepsins D and B, and GCase in live neurons cultured in microfluidic devices that allow for physical and fluidic separation of axons from the soma/dendrites. Our study demonstrates that active lysosomes are delivered from the soma to distal axons and growth cones, where they fuse with autophagosomes and perform local substrate degradation. Thus, we are now well-positioned to extend this work to provide some of the first insights into the regulation of transport, distribution, and degradation capacity of axonal lysosomes in health and LSDs. Aim 2. Mechanistic contribution of defective autophagic transport to dopaminergic neuron (DA) degeneration. Axonopathy is an early event preceding the loss of DA neurons. However, the mechanisms underlying axonopathy are largely unknown. DA neurons have complex axon structures projecting from SNpc to the striatum; defective axonal transport and impaired autophagy-lysosomal system disturb axonal homeostasis and thus contribute to PD-linked pathogenesis, including impaired clearance of damaged organelles and Lewy aggregates, where -synuclein is a major constituent. Once delivered to the lysosome, -synuclein is degraded primarily by cathepsin D, which is reduced in DA neurons in human PD brains. Reduced lysosome degradation and increased toxicity of -synuclein contribute to the risk of PD. Disrupted autophagy is also associated with presynaptic accumulation of -synuclein and LRRK2. Our recent study reveals a new motor-adaptor sharing mechanism underlying dynein-driven and snapin-mediated transport of autophagosome from distal axons toward the soma (Cheng et al., JCB 2015). Autophagosomes gain such ride-on service by sharing LE-loaded transport machinery following their fusion. This pathway ensures neurons effectively remove autophagosomes generated in distal axons. Given the critical role of snapin in mediating retrograde transport in neurons (Cai et al., Neuron 2010), we generated a DA-targeted snapin cKO mouse by crossing snapinloxp/loxp with DAT-cre mice. We are testing our hypothesis that impaired retrograde transport of axonal autophagosomes is one of the PD-linked mechanisms underlying autophagic stress, synucleinopathy, axonopathy, and DA neuron loss. Thus, snapin cKO mice provide an ideal model to test our hypothesis. Aim 3: Mechanistic contribution of defective presynaptic cargo transport to the autism-linked pathogenesis. The formation of new synapses and maintenance and remodeling of mature synapses require targeted delivery of newly synthesized presynaptic cargoes from the soma to synapses. Among these presynaptic components, scaffolding proteins Piccolo and Bassoon function as organizers of the active zone and appear the earliest at newly formed presynaptic terminals. Piccolo-Bassoon transport vesicles (PTVs) undergo axonal transport in precursor organelles containing multiple presynaptic proteins. Regulation of PTV transport ensures that active zone proteins are delivered to synapses. However, it remains unclear whether impaired PTV transport contributes to neurodevelopmental disorders such as autism spectrum disorders (ASDs). ASDs are characterized by impaired social interactions, communication deficits, and repetitive behaviors. Recent studies have suggested that some ASD-linked genes associate with synapse formation during brain development, supporting the hypothesis that dysregulation of synapse formation, maintenance, and function is a key factor in ASD pathophysiology. We previously identified syntabulin as a kinesin-1 (KIF5) motor adaptor that mediates axonal transport of PTV cargos to synapses (Su et al., Nature Cell Biology 2004). Knockdown of syntabulin reduces axonal delivery of presynaptic components, impairing synaptic formation in developing neurons and synaptic remodeling in mature neurons (Cai et al., JNS 2007; Ma et al., JNS 2009). Consistently, expressing syntabulin mutant defective in binding KIF5 motors reduces PTV cargo density in distal axons. These findings suggest that syntabulin-mediated transport is critical for synaptic formation and maintenance. A recent genetic study of autism patients identified a de-novo syntabulin variant that abolishes its interaction with KIF5. Thus, there is an urgent need to establish axonal transport and presynaptic mechanisms underlying autism-associated phenotypes. Using syntabulin cKO mice and an autism-linked syntabulin de-novo mutation, we propose to investigate whether defects in presynaptic cargo transport serve as a new mechanism contributing to the pathogenesis of autism. These studies will establish whether defective presynaptic cargo transport is associated with behavioral abnormalities in mice that bear similarities to human autism patients. Publications: Su, Q*., Q. Cai*, C. Gerwin, C. L. Smith, and Z.-H. Sheng (2004). Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Cai, Q., P.-Y. Pan, and Z.-H. Sheng (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Ma, H., Q. Cai, W. Lu, Z.-H. Sheng, and S. Mochida (2009). KIF5 motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons. Journal of Neuroscience 29, 13019-13029. Cai, Q., L. Lu, J.-H. Tian, Y.-B. Zhu, H. Qiao, and Z.-H. Sheng (2010). Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons. Neuron 68, 73-86. Cheng, X.-T. B. Zhou, M.-Y. Lin, Q. Cai, and Z.-H. Sheng (2015). Axonal autophagosomes acquire dynein motors for retrograde transport through fusion with late endosomes. Journal of Cell Biology 209, 377-386. Di Giovanni, J. and Z.-H. Sheng (2015). Regulation of synaptic activity by snapin-mediated endo-lysosomal transport and sorting. EMBO J 34, 2059-2077. Xie*, Y., B. Zhou*, M.-Y. Lin, et al., and Z.-H. Sheng (2015). Endo-lysosome deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS-linked mice. Neuron 87, 355-370. Cheng X-T, Xie Y, Zhou B, Huang N, Farfel-Becker T & Sheng Z-H (2018). Characterization of LAMP1-labeled non-degradative lysosomal and endocytic compartments in nervous systems. Journal of Cell Biology (Epub